Phenothiazine kinesin inhibitors

ABSTRACT

Phenothiazine derivatives of formula (I) are disclosed. The compounds are inhibitors of the mitotic kinesin KSP and are useful in the treatment of cellular proliferative diseases, such as cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Ser.No. 60/263,092, filed Jan. 19, 2001, which is incorporated by referencein their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to phenothiazine derivatives which are inhibitorsof the mitotic kinesin KSP and are useful in the treatment of cellularproliferative diseases, for example cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation.

BACKGROUND OF THE INVENTION

Among the therapeutic agents used to treat cancer are the taxanes andvinca alkaloids, which act on microtubules. Microtubules are the primarystructural element of the mitotic spindle. The mitotic spindle isresponsible for distribution of replicate copies of the genome to eachof the two daughter cells that result from cell division. It is presumedthat disruption of the mitotic spindle by these drugs results ininhibition of cancer cell division, and induction of cancer cell death.However, microtubules form other types of cellular structures, includingtracks for intracellular transport in nerve processes. Because theseagents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits which would berealized if the side effects associated with the administration of theseagents could be reduced. Traditionally, dramatic improvements in thetreatment of cancer are associated with identification of therapeuticagents acting through novel mechanisms. Examples of this include notonly the taxanes, but also the camptothecin class of topoisomerase Iinhibitors. From both of these perspectives, mitotic kinesins areattractive targets for new anti-cancer agents.

Mitotic kinesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic kinesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of ATP into mechanicalforce which drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

Among the mitotic kinesins which have been identified is KSP. KSPbelongs to an evolutionarily conserved kinesin subfamily of plusend-directed microtubule motors that assemble into bipolar homotetramersconsisting of antiparallel homodimers. During mitosis KSP associateswith microtubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described (Blangy, et al., Cell,83:1159–69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635–42(1996); Galgio et al., J. Cell Biol., 135:339–414 (1996); Blangy, etal., J Biol. Chem., 272:19418–24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174–82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551–61 (1998); Kaiser, et al., JBC 274:18925–31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426), and a fragment of theKSP gene (TRIP5) has been described (Lee, et al., Mol Endocrinol.,9:243–54 (1995); GenBank accession number L40372). Xenopus KSP homologs(Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported.

Mitotic kinesins are attractive targets for the discovery anddevelopment of novel mitotic chemotherapeutics. Accordingly, it is anobject of the present invention to provide methods and compositionsuseful in the inhibition of KSP, a mitotic kinesin.

Phenothiazines have been known as psychopharmacologic agents for manyyears. Chlorpromazine, fluphenazine, perphenazine, trifluoperazine,promazine and thioridazine are typical examples. Inhibition of KSP byphenothiazines has not been described.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides compositions and methods that can be used to treat diseases ofproliferating cells. The compositions are KSP inhibitors, particularlyhuman KSP inhibitors.

In one aspect, the invention relates to methods for treating cellularproliferative diseases, for treating disorders associated with KSPkinesin activity, and for inhibiting KSP kinesin. The methods employcompounds of the formula:

wherein

-   R¹ is hydrogen, halogen or CF₃;-   R² is chosen from hydrogen and lower alkyl;-   R³ is hydrogen;-   R⁴ and R⁵ are independently chosen from hydrogen, alkyl, substituted    alkyl, alkylaryl, substituted alkylaryl, alkylheteroaryl and    substituted alkylheteroaryl; or-   any of R², R³ and R⁴ taken together with the intervening atoms form    one or more five- to seven-membered rings, or a pharmaceutically    acceptable salt thereof.

The ring may be substituted with one or more alkyl, aryl, alkoxy, halo,alkylaryl or substituted alkylaryl substituents. It is necessary foractivity that the phenothiazine contain at least one five- toseven-membered ring in addition to the three rings of the phenothiazine.

Diseases and disorders that respond to therapy with compounds of theinvention include cancer, hyperplasia, restenosis, cardiac hypertrophy,immune disorders and inflammation.

In another aspect, the invention relates to compounds useful ininhibiting KSP kinesin. The compounds have the structures shown above.

In an additional aspect, the present invention provides methods ofscreening for compounds that will bind to a KSP kinesin, for examplecompounds that will displace or compete with the binding of thecompositions of the invention. The methods comprise combining a labeledcompound of the invention, a KSP kinesin, and at least one candidateagent and determining the binding of the candidate bioactive agent tothe KSP kinesin.

In a further aspect, the invention provides methods of screening formodulators of KSP kinesin activity. The methods comprise combining acomposition of the invention, a KSP kinesin, and at least one candidateagent and determining the effect of the candidate bioactive agent on theKSP kinesin activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a class of novel phenothiazinesthat are modulators of mitotic kinesins. By inhibiting or modulatingmitotic kinesins, but not other kinesins (e.g., transport kinesins),specific inhibition of cellular proliferation is accomplished. Thus, thepresent invention capitalizes on the finding that perturbation ofmitotic kinesin function causes malformation or dysfunction of mitoticspindles, frequently resulting in cell cycle arrest and cell death. Themethods of inhibiting a human KSP kinesin comprise contacting aninhibitor of the invention with a KSP kinesin, particularly human KSPkinesins, including fragments and variants of KSP. The inhibition can beof the ATP hydrolysis activity of the KSP kinesin and/or the mitoticspindle formation activity, such that the mitotic spindles aredisrupted. Meiotic spindles may also be disrupted.

An object of the present invention is to develop inhibitors andmodulators of mitotic kinesins, in particular KSP, for the treatment ofdisorders associated with cell proliferation. Traditionally, dramaticimprovements in the treatment of cancer, one type of cell proliferativedisorder, have been associated with identification of therapeutic agentsacting through novel mechanisms. Examples of this include not only thetaxane class of agents that appear to act on microtubule formation, butalso the camptothecin class of topoisomerase I inhibitors. Thecompositions and methods described herein can differ in theirselectivity and are preferably used to treat diseases of proliferatingcells, including, but not limited to cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation.

Accordingly, the present invention relates to methods employingphenothiazines of the formula:

wherein

-   R¹ is hydrogen, halogen or CF₃;-   R¹ is chosen from hydrogen and lower alkyl;-   R³ is hydrogen;-   R⁴ and R⁵ are independently chosen from hydrogen, alkyl, substituted    alkyl, alkylaryl, substituted alkylaryl, alkylheteroaryl and    substituted alkylheteroaryl; or-   any of R², R³ and R⁴ taken together with the intervening atoms form    one or more five- to seven-membered rings that may be optionally    substituted with one or more alkyl, aryl, alkoxy, halo, alkylaryl or    substituted alkylaryl substituents, or a pharmaceutically acceptable    salt thereof. It is necessary for activity that the phenothiazine    contain at least one five- to seven-membered ring in addition to the    three rings of the phenothiazine.

All of the compounds falling within the foregoing parent genus and itssubgenera are useful as kinesin inhibitors, but not all the compoundsare novel. In particular, certain known species fall within the genus inwhich R⁴ and R⁵ have the full breadth of operative substituents,although no utility in inhibiting kinesin has been suggested for thesespecies. Any narrowing of the claims or specific exceptions that mightbe added to these claims reflect applicants' intent to avoid claimingsubject matter that, while functionally part of the inventive concept,is not patentable to them for reasons having nothing to do with thescope of their invention. In particular, the novel compounds that arethe subject of the claims are described by the formula:

wherein R¹, R² and R³ are as defined above;

-   R^(4a) is chosen from hydrogen and lower alkyl; and-   R^(5a) is chosen from alkylaryl, substituted alkylaryl,    alkylheteroaryl and substituted alkylheteroaryl; or-   any of R², R³ and R^(4a) taken together with the intervening atoms    form one or more five- to seven-membered rings, which may be    optionally substituted with one or more alkyl, aryl, alkoxy, halo,    alkylaryl or substituted alkylaryl substituents, or a    pharmaceutically acceptable salt thereof.

Preferred compounds of the methods and compositions are those in whichR³ is hydrogen and R² and R^(4a) form a five- to seven-membered ring.Such compounds include phenothiazines of formula

In most preferred compounds R^(5a) is benzyl or substituted benzyl.

Other preferred compounds of the methods and compositions are those inwhich R² and R³ are hydrogen and R^(5a) is alkylaryl or substitutedalkylaryl, particularly those in which R^(5a) is benzyl or substitutedbenzyl.

Definitions

Alkyl is intended to include linear, branched, or cyclic hydrocarbonstructures and combinations thereof. Lower alkyl refers to alkyl groupsof from 1 to 5 carbon atoms. Examples of lower alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like.Preferred alkyl groups are those of C₂₀ or below. More preferred alkylgroups are those of C₁₃ or below. Cycloalkyl is a subset of alkyl andincludes cyclic hydrocarbon groups of from 3 to 13 carbon atoms.Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl,norbornyl, adamantyl and the like. In this application, alkyl refers toalkanyl, alkenyl and alkynyl residues; it is intended to includecyclohexylmethyl, vinyl, allyl, isoprenyl and the like. Alkylene refersto the same residues as alkyl, but having two points of attachment.Examples of alkylene include ethylene (—CH₂CH₂—), propylene(—CH₂CH₂CH₂—), dimethylpropylene (—CH₂C(CH₃)₂CH₂—) andcyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). When an alkyl residue having aspecific number of carbons is named, all geometric isomers having thatnumber of carbons are intended to be encompassed; thus, for example,“butyl” is meant to include n-butyl, sec-butyl, isobutyl and t-butyl;“propyl” includes n-propyl and isopropyl.

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of astraight, branched, cyclic configuration and combinations thereofattached to the parent structure through an oxygen. Examples includemethoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy andthe like. Lower-alkoxy refers to groups containing one to four carbons.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. Lower-acyl refers to groups containingone to four carbons.

Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromaticring containing 0–3 heteroatoms selected from O, N, or S; a bicyclic 9-or 10-membered aromatic or heteroaromatic ring system containing 0–3heteroatoms selected from O, N, or S; or a tricyclic 13- or 14-memberedaromatic or heteroaromatic ring system containing 0–3 heteroatomsselected from O, N, or S. The aromatic 6- to 14-membered carbocyclicrings include, e.g., benzene, naphthalene, indane, tetralin, andfluorene and the 5- to 10-membered aromatic heterocyclic rings include,e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole,furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,pyrazine, tetrazole and pyrazole.

Alkylaryl refers to a residue in which an aryl moiety is attached to theparent structure via an alkyl residue. Examples are benzyl, phenethyl,phenylvinyl, phenylallyl and the like. Alkylheteroaryl refers to aresidue in which a heteroaryl moiety is attached to the parent structurevia an alkyl residue. Examples include furanylmethyl, pyridinylmethyl,pyrimidinylethyl and the like.

Heterocycle means a cycloalkyl or aryl residue in which one to four ofthe carbons is replaced by a heteroatom such as oxygen, nitrogen orsulfur. Examples of heterocycles that fall within the scope of theinvention include imidazoline, pyrrolidine, pyrazole, pyrrole, indole,quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran,benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl,when occurring as a substituent), tetrazole, morpholine, thiazole,pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline,isoxazole, dioxane, tetrahydrofuran and the like. Examples ofsubstituted heterocyclyl include 4-methyl-1-piperazinyl and4-benzyl-1-piperidinyl.

Substituted alkyl, aryl and heteroaryl or heterocyclyl refer to alkyl,aryl, heteroaryl or heterocyclyl wherein H atoms are replaced withalkyl, halogen, hydroxy, alkoxy, alkylenedioxy (e.g. methylenedioxy)fluoroalkyl, carboxy (—COOH), carboalkoxy (i.e. acyloxy RCOO—),carboxyalkyl (—COOR), carboxamido, sulfonamidoalkyl, sulfonamidoaryl,aminocarbonyl, benzyloxycarbonylamino (CBZ-amino), cyano, carbonyl,nitro, dialkylamino, alkylamino, amino, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylsulfonamido, arylthio, arylsulfinyl, arylsulfonyl,amidino, phenyl, benzyl, heteroaryl, heterocyclyl, phenoxy, benzyloxy,or heteroaryloxy. For the purposes of the present invention, substitutedalkyl also includes oxaalkyl residues, i.e. alkyl residues in which oneor more carbons has been replaced by oxygen.

Halogen refers to fluorine, chlorine, bromine or iodine. Fluorine,chlorine and bromine are preferred.

Many of the compounds described herein contain one or more asymmetriccenters (e.g. the carbons to which R² and R³ are attached) and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)- or(S)- . The present invention is meant to include all such possibleisomers, including racemic mixtures, optically pure forms andintermediate mixtures. Optically active (R)- and (S)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included.

When desired, the R- and S-isomers may be resolved by methods known tothose skilled in the art, for example by formation of diastereoisomericsalts or complexes which may be separated, for example, bycrystallisation; via formation of diastereoisomeric derivatives whichmay be separated, for example, by crystallisation, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic oxidation orreduction, followed by separation of the modified and unmodifiedenantiomers; or gas-liquid or liquid chromatography in a chiralenvironment, for example on a chiral support, such as silica with abound chiral ligand or in the presence of a chiral solvent. It will beappreciated that where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be required to liberate the desired enantiomeric form.Alternatively, specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

The graphic representations of racemic, ambiscalemic and scalemic orenantiomerically pure compounds used herein are taken from Maehr J.Chem. Ed. 62, 114–120 (1985): solid and broken wedges are used to denotethe absolute configuration of a chiral element; wavy lines indicatedisavowal of any stereochemical implication which the bond it representscould generate; solid and broken bold lines are geometric descriptorsindicating the relative configuration shown but denoting racemiccharacter; and wedge outlines and dotted or broken lines denoteenantiomerically pure compounds of indeterminate absolute configuration.

In some embodiments, two R groups may be joined to form a ringstructure. Again, the ring structure may contain heteroatoms and may besubstituted with one or more substituents.

The compositions of the invention are synthesized as outlined below,utilizing techniques well known in the art. Once made, the compositionsof the invention find use in a variety of applications. As will beappreciated by those in the art, mitosis may be altered in a variety ofways; that is, one can affect mitosis either by increasing or decreasingthe activity of a component in the mitotic pathway. Stated differently,mitosis may be affected (e.g., disrupted) by disturbing equilibrium,either by inhibiting or activating certain components. Similarapproaches may be used to alter meiosis.

In a preferred embodiment, the compositions of the invention are used tomodulate mitotic spindle formation, thus causing prolonged cell cyclearrest in mitosis. By “modulate” herein is meant altering mitoticspindle formation, including increasing and decreasing spindleformation. By “mitotic spindle formation” herein is meant organizationof microtubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

The compositions of the invention are useful to bind to and/or modulatethe activity of a mitotic kinesin, KSP. In a preferred embodiment, theKSP is human KSP, although KSP kinesins from other organisms may also beused. In this context, modulate means either increasing or decreasingspindle pole separation, causing malformation, i.e., splaying, ofmitotic spindle poles, or otherwise causing morphological perturbationof the mitotic spindle. Also included within the definition of KSP forthese purposes are variants and/or fragments of KSP. See U.S. PatentApplication “Methods of Screening for Modulators of Cell Proliferationand Methods of Diagnosing Cell Proliferation States”, filed Oct. 27,1999 (U.S. Ser. No. 09/428,156), hereby incorporated by reference in itsentirety. In addition, other mitotic kinesins may be used in the presentinvention. However, the compositions of the invention have been shown tohave specificity for KSP.

For assay of activity, generally either KSP or a compound according tothe invention is non-diffusably bound to an insoluble support havingisolated sample receiving areas (e.g., a microtiter plate, an array,etc.). The insoluble support may be made of any composition to which thecompositions can be bound, is readily separated from soluble material,and is otherwise compatible with the overall method of screening. Thesurface of such supports may be solid or porous and of any convenientshape. Examples of suitable insoluble supports include microtiterplates, arrays, membranes and beads. These are typically made of glass,plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose,Teflon™, etc. Microtiter plates and arrays are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples. The particular manner ofbinding of the composition is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies (which do not sterically blockeither the ligand binding site or activation sequence when the proteinis bound to the support), direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or agent, excess unboundmaterial is removed by washing. The sample receiving areas may then beblocked through incubation with bovine serum albumin (BSA), casein orother innocuous protein or other moiety.

The antimitotic agents of the invention may be used on their own tomodulate the activity of a mitotic kinesin, particularly KSP. In thisembodiment, the mitotic agents of the invention are combined with KSPand the activity of KSP is assayed. Kinesin activity is known in the artand includes one or more kinesin activities. Kinesin activities includethe ability to affect ATP hydrolysis; microtubule binding; gliding andpolymerization/depolymerization (effects on microtubule dynamics);binding to other proteins of the spindle; binding to proteins involvedin cell-cycle control; serving as a substrate to other enzymes; such askinases or proteases; and specific kinesin cellular activities such asspindle pole separation.

Methods of performing motility assays are well known to those of skillin the art. (See e.g., Hall, et al. (1996), Biophys. J., 71: 3467–3476,Turner et al., 1996, Anal. Biochem. 242 (1):20–5; Gittes et al., 1996,Biophys. J. 70(1): 418–29; Shirakawa et al., 1995, J. Exp. Biol. 198:1809–15; Winkelmann et al., 1995, Biophys. J. 68: 2444–53; Winkelmann etal., 1995, Biophys. J. 68: 72S.)

Methods known in the art for determining ATPase hydrolysis activity alsocan be used. Preferably, solution based assays are utilized. U.S.application Ser. No. 09/314,464, filed May 18, 1999, hereby incorporatedby reference in its entirety, describes such assays. Alternatively,conventional methods are used. For example, P_(i) release from kinesincan be quantified. In one preferred embodiment, the ATPase hydrolysisactivity assay utilizes 0.3 M PCA (perchloric acid) and malachite greenreagent (8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate,and 0.8 mM Triton X-1 00). To perform the assay, 10 μL of reaction isquenched in 90 μL of cold 0.3 M PCA. Phosphate standards are used sodata can be converted to mM inorganic phosphate released. When allreactions and standards have been quenched in PCA, 100 μL of malachitegreen reagent is added to the relevant wells in e.g., a microtiterplate. The mixture is developed for 10–15 minutes and the plate is readat an absorbance of 650 nm. If phosphate standards were used, absorbancereadings can be converted to mM P_(i) and plotted over time.Additionally, ATPase assays known in the art include the luciferaseassay.

ATPase activity of kinesin motor domains also can be used to monitor theeffects of modulating agents. In one embodiment ATPase assays of kinesinare performed in the absence of microtubules. In another embodiment, theATPase assays are performed in the presence of microtubules. Differenttypes of modulating agents can be detected in the above assays. In apreferred embodiment, the effect of a modulating agent is independent ofthe concentration of microtubules and ATP. In another embodiment, theeffect of the agents on kinesin ATPase can be decreased by increasingthe concentrations of ATP, microtubules or both. In yet anotherembodiment, the effect of the modulating agent is increased byincreasing concentrations of ATP, microtubules or both.

Agents that modulate the biochemical activity of KSP in vitro may thenbe screened in vivo. Methods for such agents in vivo include assays ofcell cycle distribution, cell viability, or the presence, morphology,activity, distribution, or amount of mitotic spindles. Methods formonitoring cell cycle distribution of a cell population, for example, byflow cytometry, are well known to those skilled in the art, as aremethods for determining cell viability. See for example, U.S. PatentApplication “Methods of Screening for Modulators of Cell Proliferationand Methods of Diagnosing Cell Proliferation States,” filed Oct. 22,1999, Ser. No. 09/428,156, hereby incorporated by reference in itsentirety.

In addition to the assays described above, microscopic methods formonitoring spindle formation and malformation are well known to those ofskill in the art (see, e.g., Whitehead and Rattner (1998), J. Cell Sci.111:2551–61; Galgio et al, (1996) J. Cell biol., 135:399–414).

The compositions of the invention inhibit the KSP kinesin. One measureof inhibition is IC₅₀, defined as the concentration of the compositionat which the activity of KSP is decreased by fifty percent. Preferredcompositions have IC₅₀'s of less than about 1 mM, with preferredembodiments having IC₅₀'s of less than about 100 μM, with more preferredembodiments having IC₅₀'s of less than about 10 μM, with particularlypreferred embodiments having IC₅₀'s of less than about 1 μM, andespecially preferred embodiments having IC₅₀'s of less than about 500nM. Measurement of IC₅₀ is done using an ATPase assay.

Another measure of inhibition is K_(i). For compounds with IC₅₀'s lessthan 1 μM, the K_(i) or K_(d) is defined as the dissociation rateconstant for the interaction of the phenothiazine with KSP. Preferredcompounds have K_(i)'s of less than about 100 μM, with preferredembodiments having K_(i)'s of less than about 10 μM, and particularlypreferred embodiments having K_(i)'s of less than about 1 μM andespecially preferred embodiments having K_(i)'s of less than about 500nM.

Another measure of inhibition is GI₅₀, defined as the concentration ofthe compound that results in a decrease in the rate of cell growth byfifty percent. Preferred compounds have GI₅₀'s of less than about 1 mM.The level of preferability of embodiments is a function of their GI₅₀:those having GI₅₀'s of less than about 20 μM are more preferred; thosehaving GI₅₀'s of 10 μM more so; those having GI₅₀ of less than about 1μM more so; those having GI₅₀'s of 500 nM more so. Measurement of GI₅₀is done using a cell proliferation assay.

The compositions of the invention are used to treat cellularproliferation diseases. Disease states which can be treated by themethods and compositions provided herein include, but are not limitedto, cancer (further discussed below), autoimmune disease, arthritis,graft rejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. It is appreciated that in some cases the cells may not bein a hyper or hypo proliferation state (abnormal state) and stillrequire treatment. For example, during wound healing, the cells may beproliferating “normally”, but proliferation enhancement may be desired.Similarly; as discussed above, in the agriculture arena, cells may be ina “normal” state, but proliferation modulation may be desired to enhancea crop by directly enhancing growth of a crop, or by inhibiting thegrowth of a plant or organism which adversely affects the crop. Thus, inone embodiment, the invention herein includes application to cells orindividuals afflicted or impending affliction with any one of thesedisorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma (pinealoma), glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia (acuteand chronic), acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignantlymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above identified conditions.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant administration of atherapeutically effective dose of the mitotic agents of the invention toa cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantcells in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Mitotic agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1–100 wt. %. The agents may be administered alone orin combination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts. “Pharmaceuticallyacceptable acid addition salt” refers to those salts that retain thebiological effectiveness of the free bases and that are not biologicallyor otherwise undesirable, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The administration of the mitotic agents of the present invention can bedone in a variety of ways as discussed above, including, but not limitedto, orally, subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the anti-mitotic agents may be directly appliedas a solution or spray.

To employ the compounds of the invention in a method of screening forcompounds that bind to KSP kinesin, the KSP is bound to a support, and acompound of the invention (which is a mitotic agent) is added to theassay. Alternatively, the compound of the invention is bound to thesupport and KSP is added. Classes of compounds among which novel bindingagents may be sought include specific antibodies, non-natural bindingagents identified in screens of chemical libraries, peptide analogs,etc. Of particular interest are screening assays for candidate agentsthat have a low toxicity for human cells. A wide variety of assays maybe used for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, functional assays (phosphorylation assays, etc.) andthe like.

The determination of the binding of the mitotic agent to KSP may be donein a number of ways. In a preferred embodiment, the mitotic agent (thecompound of the invention) is labeled, for example, with a fluorescentor radioactive moiety and binding determined directly. For example, thismay be done by attaching all or a portion of KSP to a solid support,adding a labeled mitotic agent (for example a compound of the inventionin which at least one atom has been replaced by a detectable isotope),washing off excess reagent, and determining whether the amount of thelabel is that present on the solid support. Various blocking and washingsteps may be utilized as is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, orwith fluorophores. Alternatively, more than one component may be labeledwith different labels; using ¹²⁵I for the proteins, for example, and afluorophor for the mitotic agents.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates. “Candidate bioactive agent” or “drugcandidate” or grammatical equivalents as used herein describe anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, etc., to be tested for bioactivity. Theymay be capable of directly or indirectly altering the cellularproliferation phenotype or the expression of a cellular proliferationsequence, including both nucleic acid sequences and protein sequences.In other cases, alteration of cellular proliferation protein bindingand/or activity is screened. Screens of this sort may be performedeither in the presence or absence of microtubules. In the case whereprotein binding or activity is screened, preferred embodiments excludemolecules already known to bind to that particular protein, for example,polymer structures such as microtubules, and energy sources such as ATP.Preferred embodiments of assays herein include candidate agents which donot bind the cellular proliferation protein in its endogenous nativestate termed herein as “exogenous” agents. In another preferredembodiment, exogenous agents further exclude antibodies to KSP.

Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl,hydroxyl, ether, or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Competitive screening assays may be done by combining KSP and a drugcandidate in a first sample. A second sample comprises a mitotic agent,KSP and a drug candidate. This may be performed in either the presenceor absence of microtubules. The binding of the drug candidate isdetermined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to KSP and potentially modulating its activity. That is, if thebinding of the drug candidate is different in the second sample relativeto the first sample, the drug candidate is capable of binding to KSP.

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to KSP,such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to KSP for a timesufficient to allow binding, if present. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C.

Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high throughput screening. Typicallybetween 0.1 and 1 hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to KSP and thus is capable of binding to, andpotentially modulating, the activity of KSP. In this embodiment, eithercomponent can be labeled. Thus, for example, if the competitor islabeled, the presence of label in the wash solution indicatesdisplacement by the agent. Alternatively, if the candidate agent islabeled, the presence of the label on the support indicatesdisplacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound toKSP with a higher affinity. Thus, if the candidate agent is labeled, thepresence of the label on the support, coupled with a lack of competitorbinding, may indicate the candidate agent is capable of binding to KSP.

It may be of value to identify the binding site of KSP. This can be donein a variety of ways. In one embodiment, once KSP has been identified asbinding to the mitotic agent, KSP is fragmented or modified and theassays repeated to identify the necessary components for binding.

Modulation is tested by screening for candidate agents capable ofmodulating the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. Thus, in this embodiment, the candidateagent should both bind to KSP (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods and in vivo screening ofcells for alterations in cell cycle distribution, cell viability, or forthe presence, morpohology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drugcandidates that bind to the native KSP, but cannot bind to modified KSP.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES

Abbreviations and Definitions

The following abbreviations and terms have the indicated meaningsthroughout:

-   Ac=acetyl-   BNB=4-bromomethyl-3-nitrobenzoic acid-   Boc=t-butyloxy carbonyl-   Bu=butyl-   c-=cyclo-   CBZ=carbobenzoxy=benzyloxycarbonyl-   DBU=diazabicyclo[5.4.0]undec-7-ene-   DCM=dichloromethane=methylene chloride=CH₂Cl₂-   DCE=dichloroethane-   DEAD=diethyl azodicarboxylate-   DIC=diisopropylcarbodiimide-   DIEA=N,N-diisopropylethylamine-   DMAP=4-N,N-dimethylaminopyridine-   DMF=N,N-dimethylformamide-   DMSO=dimethyl sulfoxide-   DVB=1,4-divinylbenzene-   EDCI=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-   EEDQ=2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline-   Et=ethyl-   Fmoc=9-fluorenylmethoxycarbonyl-   GC=gas chromatography-   HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HMDS=hexamethyldisilazane-   HOAc=acetic acid-   HOBt=hydroxybenzotriazole-   Me=methyl-   mesyl=methanesulfonyl-   MTBE=methyl t-butyl ether-   NMO=N-methylmorpholine oxide-   PEG=polyethylene glycol-   Ph=phenyl-   PhOH=phenol-   PfP=pentafluorophenol-   PPTS=pyridinium p-toluenesulfonate-   Py=pyridine-   PyBroP=bromo-tris-pyrrolidino-phosphonium hexafluorophosphate-   rt=room temperature-   sat=d=saturated-   s-=secondary-   t-=tertiary-   TBDMS=t-butyldimethylsilyl-   TES=triethylsilane-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran-   TMOF=trimethyl orthoformate-   TMS=trimethylsilyl-   tosyl=p-toluenesulfonyl-   Trt=triphenylmethyl    Synthesis of Compounds

The syntheses of several prototypical phenothiazines are shown below.Other phenothiazines are made in analogous fashion:Phenothiazine Synthesis—Procedure A

Phenothiazine Synthesis—Procedure B

Phenothiazine Procedure A

Synthesis of Benzyl-[2-(2-chloro-phenothiazin-10-yl)-ethyl]-methylamineFumarate.

Synthesis of (2-chlorophenothiazin-10-yl)acetic Acid

2-Chloro-10H-phenothiazine (10.0 g, 42.8 mmol) was dissolved in THF (100mL) and DMSO (10 mL). Sodium hydride (1.0 g, 43.4 mmol) was added, andthe mixture was heated to reflux until gas evolution ceased. Ethylbromoacetate (10 g, 59.9 mmol) was added slowly via syringe. The mixturewas heated at reflux for 12 h. Sodium hydride (1.0 g, 43.4 mmol) andethyl bromoacetate (5 g, 29.9 mmol) were added, and the mixture washeated an additional 12 h. The mixture was cooled to room temperatureand carefully diluted with water followed by ethyl acetate. The layerswere separated, and the aqueous layer extracted with ethyl acetate. Theorganic layers were combined, dried (Na₂SO₄), filtered, and concentratedto give a dark purple oil (11.8 g). The oil was dissolved in methanol(300 mL), and aqueous sodium hydroxide (6 N, 22 mL, 132 mmol) was added.The mixture was heated to reflux for 4 h. The mixture was cooled to roomtemperature, and the solvent removed under vacuum. The residue wasdissolved in water and was extracted with diethyl ether (ether layersdiscarded). The layers were separated, and the pH of the aqueous layerwas made acidic with concentrated hydrochloric acid. The water layer wasextracted with methylene chloride. The organic layers were combined,dried (Na₂SO₄), filtered, and concentrated to give(2-chlorophenothiazin-10-yl)acetic acid (7.38 g, 56%) as a brown solid.

Synthesis of N-Benzyl-2-(2-chlorophenothiazin-10-yl)-N-methylacetamide

(2-Chlorophenothiazin-10-yl)acetic acid (480 mg, 1.65 mmol) wasdissolved in THF (10 mL). To this solution,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.58 g,8.25 mmol), 1-hydroxybenzotriazole (220 mg, 1.65 mmol), and N-methylbenzyl amine (2.0 mL, 16.5 mmol) were added. The mixture was stirred atroom temperature overnight and was then diluted with water and methylenechloride. The aqueous layer was made basic with sodium hydroxide (1N),and the layers were separated. The aqueous layer was extracted withmethylene chloride. The organic layers were combined, washed with waterand brine, dried (Na₂SO₄), filtered, and concentrated to provide amixture of N-methyl benzyl amine and product (3.8 g). The residue waspurified with a plug of SiO₂. The product was recrystallized from hexaneand ethyl acetate to provideN-benzyl-2-(2-chlorophenothiazin-10-yl)-N-methylacetamide as a whitesolid (258 mg, 40%).

Synthesis of Benzyl-[2-(2-chlorophenothiazin-10-yl)-ethyl]methyl AmineFumarate

N-Benzyl-2-(2-chlorophenothiazin-10-yl)-N-methylacetamide (140 mg, 0.355mmol) was dissolved in THF (5 mL). A solution of borane• THF complex inTHF (1.0 M, 5 mL, 5 mmol) was added, and the solution heated to refluxfor 4 h. The mixture was carefully diluted with saturated HCl inmethanol and stirred for 30 min. The solvent was removed under vacuum,and the residue dissolved in ethyl acetate and aqueous sodium hydroxide(1 N). The layers were separated and the aqueous layer extracted withethyl acetate. The organic layers were combined, dried (Na₂SO₄),filtered, and concentrated to providebenzyl-[2-(2-chlorophenothiazin-10-yl)-ethyl]methyl amine as a whitesolid (160 mg). The crude product was purified by SiO₂ chromatography toprovide pure product (50 mg, 34%) and 100 mg of impure product. The pureproduct was dissolved in acetone and a solution of fumaric acid (0.8 mL,0.02 g/mL in methanol) was added. The solvents were removed undervacuum, and the residue slurried in chloroform and filtered to providethe sub-titled compound (47 mg) as an off-white solid.

Phenothiazine Procedure B

Synthesis of2-Chloro-10-(1-pyridin-3-ylmethylpiperidin-3-yl)-10H-phenothiazineHydrochloride

Synthesis of 2,4-dichloro-2′-nitrodiphenyl Thioether

2,4-Dichlorobenzenethiol (16.1 g, 89.9 mmol) was dissolved in ethanol(200 mL) and was heated to reflux. A solution of sodium acetate (11.1 g,135 mmol) dissolved in 100 mL ethanol was added, followed by a solutionof 1-iodo-2-nitrobenzene (33.6 g, 135 mmol) in ethanol (100 mL, added insmall portions over 5 min). The mixture was heated at reflux for 12 h.The mixture was cooled to room temperature and concentrated undervacuum. The residue was dissolved in water and methylene chloride, andthe aqueous layer was diluted with saturated sodium bicarbonate. Thelayers were separated, and the aqueous layer was extracted withmethylene chloride. The organic layers were combined, dried (Na₂SO₄),filtered, and concentrated to provide a solid. The residue was washedwith 30% ethanol in water (300 mL) and collected by vacuum filtration.The filter cake was rinsed with 30% ethanol in water. The solid wastreated with methanol (100 mL), and the product collected by filtrationto provide 2,4-dichloro-2′-nitrodiphenyl thioether (25.6 g, 94%) as ayellow solid.

Synthesis of 2,4-dichloro-2′-aminodiphenyl Thioether

2,4-Dichloro-2′-nitrodiphenyl thioether (22.4 g, 75 mmol) was dissolvedin ethyl acetate (125 mL) at 40° C. Adams catalyst (PtO₂, 2.5 g) wasadded, and hydrogen was vigorously bubbled through the solution for 1 h.The mixture was stirred overnight under a static atmosphere of hydrogen.The mixture was cooled to room temperature and filtered through a bed ofcellulose. The solvent was removed under vacuum to provide2,4-dichloro-2′-aminodiphenyl thioether (18.1 g, 89%) as a yellow oil.

Synthesis of 3-Hydroxypiperidine-1-carboxylic Acid Tert-butyl Ester

3-Hydroxypiperidine (10.0 g, 98.9 mmol) was dissolved in methylenechloride (100 mL) and cooled to 0° C. Di-tert-butyl dicarbonate (27.3 g,125 mmol) was added at once (copious gas evolution was observed). Themixture was stirred for 12 h at room temperature. The solvent wasremoved under vacuum to provide 3-hydroxypiperidine-1-carboxylic acidtert-butyl ester (19.8 g, 100%).

Synthesis of 3-Oxopiperidine-1-carboxylic Acid Tert-butyl Ester

A mixture of DMSO (7.8 mL, 109.3 mmol) and methylene chloride (100 mL)was cooled to −78° C. Oxalyl chloride (4.8 mL, 54.7 mmol) was added tothis solution via syringe. The mixture was stirred at −78° C. for 30min. A solution of 3-hydroxypiperidine-1-carboxylic acid tert-butylester (10.0 g, 49.7 mmol) in methylene chloride (30 mL) was addeddropwise to this solution (temperature remained below −70° C.). Themixture was stirred for 30 min. Triethylamine (28 mL, 200 mmol) wasadded dropwise over 20 min. The mixture was stirred at −78° C. for 1 hand was allowed to warm to room temperature and stirred for 45 min. Themixture was diluted with water, and the layers separated. The organiclayer was washed with water, dried Na₂SO₄), filtered, and concentratedto give 3-oxopiperidine-1-carboxylic acid tert-butyl ester (10.2 g) as ayellow, brown liquid.

Synthesis of3-[2-(2,4-Dichlorophenylsulfanyl)phenylamino]piperidine-1-carboxylicAcid Tert-butyl Ester

2,4-Dichloro-2′-aminodiphenyl thioether (10.3 g, 38.1 mmol) wasdissolved in dichloroethane (120 mL). To this solution, a solution of3-oxopiperidine-1-carboxylic acid tert-butyl ester (14.0 g, 70.3 mmol)in dichloroethane (20 mL) and solid sodium triacetoxyborohydride (14.5g, 68.7 mmol) were slowly added. Acetic acid (5.4 mL, 94 mmol) was addedslowly via syringe, and the mixture stirred for 48 h. The mixture wascarefully diluted with water, and the pH was adjusted to approximately 9with aqueous sodium hydroxide (1 N). The mixture was diluted with ethylacetate, and the layers were separated. The aqueous layer was extractedwith ethyl acetate. The organic layers were combined, dried (Na₂SO₄),filtered, and concentrated to provide3-[2-(2,4-dichlorophenylsulfanyl)phenylamino]piperidine-1-carboxylicacid tert-butyl ester as a brown oil. The crude product was purified bySiO₂ chromatography resulting in pure product (8.4 g, 49%) as a lightyellow oil.

Synthesis of 3-(2-Chlorophenothiazin-10-yl)piperidine-1-carboxylic AcidTert-butyl Ester

3-[2-(2,4-Dichlorophenylsulfanyl)phenylamino]piperidine-1-carboxylicacid tert-butyl ester (8.4 g, 18.5 mmol) was dissolved in DMF (120 mL)and vigorously degassed with N₂ for 20 min. Cesium carbonate (27.2 g,83.5 mmol), copper(I) iodide (5.28 g, 27.8 mmol), and copper powder (8.4g, 1321 mmol) were added to the solution. The suspension was stirredvigorously and heated to 155–156° C. for 12 hours (N₂ was continuouslybubbled through the mixture). The mixture was cooled to room temperatureand diluted with ethyl acetate. The solids were removed by vacuumfiltration and were rinsed with ethyl acetate. The filtrate wasconcentrated under vacuum to remove 90% of the volatiles. The residuewas purified by SiO₂ chromatography to provide3-(2-chlorophenothiazin-10-yl) piperidine-1-carboxylic acid tert-butylester (4.1 g, 53%) as a white solid.

Synthesis of 2-Chloro-10-piperidin-3-yl-10H-phenothiazine Hydrochloride

3-(2-Chlorophenothiazin-10-yl) piperidine-1-carboxylic acid tert-butylester (4.1 g, 9.83 mmol) was dissolved in diethyl ether (200 mL), andthe solution cooled to 0° C. A solution of HCl in ethyl acetate (˜4 M,30 mL) was slowly added. The mixture was allowed to warn to roomtemperature and stirred for 8 h. The solvent was removed under vacuum.The residue treated with HCl in ethyl acetate (˜4 M, 75 mL) and stirredovernight. The solvent was removed under vacuum to provide2-chloro-10-piperidin-3-yl-10H-phenothiazine (3.98 g, 100%) as anoff-white solid.

Synthesis of2-Chloro-10-(1-pyridin-3-yl-methylpiperidin-3-yl)-10H-phenothiazineHydrochloride

Using methods substantially equivalent to those described in thesynthesis of3-[2-(2,4-dichloro-phenylsulfanyl)-phenylamino]-piperidine-1-carboxylicacid tert-butyl ester,

2-chloro-10-(1-pyridin-3-yl-methylpiperidin-3-yl)-10H-phenothiazine wasprepared by treatment of 2-chloro-10-piperidin-3-yl-10H-phenothiazine(240 mg, 0.757 mmol) and 3-pyridinecarboxaldehyde (81 mg, 0.757 mmol)with sodium triacetoxyborohydride (224 mg, 1.06 mmol) to provide aftertreatment with ethereal HCl, the sub-titled compound (136 mg, 41%).

Induction of Mitotic Arrest in Cell Populations Treated with aPhenothiazine KSP Inhibitor

FACS analysis to determine cell cycle stage by measuring DNA content wasperformed as follows. Skov-3 cells (human ovarian cancer) were split1:10 for plating in 10 cm dishes and grown to subconfluence with RPMI1640 medium containing 5% fetal bovine serum (FBS). The cells were thentreated with either 10 nM paclitaxel, the test compound or 0.25% DMSO(vehicle for compounds) for 24 hours. Cells were then rinsed off theplates with PBS containing 5 mM EDTA, pelleted, washed once in PBScontaining 1% FCS, and then fixed overnight in 85% ethanol at 4° C.Before analysis, the cells were pelleted, washed once, and stained in asolution of 10 μg propidium iodide and 250 μg of ribonuclease (RNAse) Aper milliliter at 37° C. for half an hour. Flow cytometry analysis wasperformed on a Becton-Dickinson FACScan, and data from 10,000 cells persample was analyzed with Modfit software.

The phenothiazine compounds, as well as the known anti-mitotic agentpaclitaxel, caused a shift in the population of cells from a G0/G1 cellcycle stage (2n DNA content) to a G2/M cell cycle stage (4n DNAcontent). Other compounds of this class were found to have similareffects.

Monopolar Spindle Formation Following Application of a Phenothiazine KSPInhibitor

To determine the nature of the G2/M accumulation, human tumor cell linesSkov-3 (ovarian), HeLa (cervical), and A549 (lung) were plated in96-well plates at densities of 4,000 cells per well (SKOV-3 & HeLa) or8,000 cells per well (A549), allowed to adhere for 24 hours, and treatedwith various concentrations of the phenothiazine compounds for 24 hours.Cells were fixed in 4% formaldehyde and stained with antitubulinantibodies (subsequently recognized using fluorescently-labeledsecondary antibody) and Hoechst dye (which stains DNA).

Visual inspection revealed that the phenothiazine compounds caused cellcycle arrest in the prometaphase stage of mitosis. DNA was condensed andspindle formation had initiated, but arrested cells uniformly displayedmonopolar spindles, indicating that there was an inhibition of spindlepole body separation. Microinjection of anti-KSP antibodies also causesmitotic arrest with arrested cells displaying monopolar spindles.

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated withPhenothiazine KSP Inhibitors

Cells were plated in 96-well plates at densities from 1000–2500cells/well of a 96-well plate (depending on the cell line) and allowedto adhere/grow for 24 hours. They were then treated with variousconcentrations of drug for 48 hours. The time at which compounds areadded is considered T₀. A tetrazolium-based assay using the reagent3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) (I.S>U.S. Pat. No. 5,185,450) (see Promega product catalog #G3580,CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay) was usedto determine the number of viable cells at T₀ and the number of cellsremaining after 48 hours compound exposure. The number of cellsremaining after 48 hours was compared to the number of viable cells atthe time of drug addition, allowing for calculation of growthinhibition.

The growth over 48 hours of cells in control wells that had been treatedwith vehicle only (0.25% DMSO) is considered 100% growth and the growthof cells in wells with compounds is compared to this. Phenothiazine KSPinhibitors inhibited cell proliferation in human tumor cell lines of thefollowing tumor types: lung (NCI-H460, A549), breast (MDA-MB-231, MCF-7,MCF-7/ADR-RES), colon (HT29, HCT15), ovarian (SKOV-3, OVCAR-3), leukemia(HL-60(TB), K-562), central nervous system (SF-268), renal (A498),osteosarcoma (U2-OS), and cervical (HeLa). In addition, a mouse tumorline (B16, melanoma) was also growth-inhibited in the presence of thephenothiazine compounds.

A Gi₅₀ was calculated by plotting the concentration of compound in μM vsthe percentage of cell growth of cell growth in treated wells. The Gi₅₀calculated for the compounds is the estimated concentration at whichgrowth is inhibited by 50% compared to control, i.e., the concentrationat which:100×[(Treated₄₈ −T ₀)/(Control₄₈ −T ₀)]=50.

All concentrations of compounds are tested in duplicate and controls areaveraged over 12 wells. A very similar 96-well plate layout and Gi₅₀calculation scheme is used by the National Cancer Institute (see Monks,et al., J. Natl. Cancer Inst. 83:757–766 (1991)). However, the method bywhich the National Cancer Institute quantitates cell number does not useMTS, but instead employs alternative methods.

Calculation of IC₅₀:

Measurement of a composition's IC₅₀ for KSP activity uses an ATPaseassay. The following solutions are used: Solution 1 consists of 3 mMphosphoenolpyruvate potassium salt (Sigma P-7127), 2 mM ATP (SigmaA-3377), 1 mM IDTT (Sigma D-9779), 5 μM paclitaxel (Sigma T-7402), 10ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2mM MgCl2 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2consists of 1 mM NADH (Sigma A8129), 0.2 mg/ml BSA (Sigma A7906),pyruvate kinase 7 U/ml, L-lactate dehydrogenase 10 U/ml (Sigma PO294),100 nM KSP motor domain, 50 μg/ml microtubules, 1 mM DTT (Sigma D9779),5 μM paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003–01), and 1 mMEGTA (Sigma E3889). Serial dilutions (8–12 two-fold dilutions) of thecomposition are made in a 96-well microtiter plate (Corning Costar 3695)using Solution 1. Following serial dilution each well has 50 μl ofSolution 1. The reaction is started by adding 50 μl of solution 2 toeach well. This may be done with a multichannel pipettor either manuallyor with automated liquid handling devices. The microtiter plate is thentransferred to a microplate absorbance reader and multiple absorbancereadings at 340 nm are taken for each well in a kinetic mode. Theobserved rate of change, which is proportional to the ATPase rate, isthen plotted as a function of the compound concentration. For a standardIC₅₀ determination the data acquired is fit by the following fourparameter equation using a nonlinear fitting program (e.g., Grafit 4):$y = {\frac{Range}{1 + \left( \frac{x}{{IC}_{50}} \right)^{s}} + {Background}}$where y is the observed rate and x the compound concentration.

The K_(i) for a compound is determined from the IC₅₀ based on threeassumptions. First, only one compound molecule binds to the enzyme andthere is no cooperativity. Second, the concentrations of active enzymeand the compound tested are known (i.e., there are no significantamounts of impurities or inactive forms in the preparations). Third, theenzymatic rate of the enzyme-inhibitor complex is zero. The rate (i.e.,compound concentration) data are fitted to the equation:$V = {V_{\max}{E_{0}\left\lbrack {I - \frac{\left( {E_{0} + I_{0} + {Kd}} \right) - \sqrt{\left( {E_{0} + I_{0} + {Kd}} \right)^{2} - {4E_{0}I_{0}}}}{2E_{0}}} \right\rbrack}}$where V is the observed rate, V_(max) is the rate of the free enzyme, I₀is the inhibitor concentration, E₀ is the enzyme concentration, andK_(d) is the dissociation constant of the enzyme-inhibitor complex.

Several representative compounds of the invention (as their fumaratesalts) were tested as described above and found to exhibit Ki's lessthan 10 μM. Their structures are as shown:

The phenothiazine compounds inhibit growth in a variety of cell lines,including cell lines (MCF-7/ADR-RES, HCT1 5) that express P-glycoprotein(also known as Multi-drug Resistance, or MDR⁺), which conveys resistanceto other chemotherapeutic drugs, such as pacilitaxel. Therefore, thephenothiazines are anti-mitotics that inhibit cell proliferation, andare not subject to resistance by overexpression of MDR⁺ bydrug-resistant tumor lines.

Compounds of this class were found to inhibit cell proliferation,although GI₅₀ values varied. GI₅₀ values for the phenothiazine compoundstested ranged from 200 nM to greater than the highest concentrationtested. By this we mean that although most of the compounds thatinhibited KSP activity biochemically did inhibit cell proliferation, forsome, at the highest concentration tested (generally about 20 μM), cellgrowth was inhibited less than 50%. Many of the compounds have GI₅₀values less than 10 μM, and several have GI₅₀ values less than 1 μM.Anti-proliferative compounds that have been successfully applied in theclinic to treatment of cancer (cancer chemotherapeutics) have GI₅₀'sthat vary greatly. For example, in A549 cells, paclitaxel GI₅₀ is 4 nM,doxorubicin is 63 nM, 5-fluorouracil is 1 μM, and hydroxyurea is 500 μM(data provided by National Cancer Institute, Developmental TherapeuticProgram, http://dtp.nci.nih.gov/). Therefore, compounds that inhibitcellular proliferation at virtually any concentration may be useful.However, preferably, compounds will have GI₅₀ values of less than 1 mM.More preferably, compounds will have GI₅₀ values of less than 20 μM.Even more preferably, compounds will have GI₅₀ values of less than 10μM. Further reduction in GI₅₀ values may also be desirable, includingcompounds with GI₅₀ values of less than 1 μM.

1. A phenothiazine of the formula

wherein R¹ is hydrogen, halogen or CF₃; R³ is hydrogen; R² and R^(4a) taken together with the intervening atoms form a five- to seven-membered rind optionally substituted with one or more alkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylaryl substituents; and R^(5a) is chosen from alkylaryl, substituted alkylaryl, alkylheteroaryl and substituted alkylheteroaryl; or a pharmaceutically acceptable salt thereof.
 2. A phenothiazine according to claim 1 of formula


3. A phenothiazine according to claim 2 wherein R^(5a) is benzyl or substituted benzyl.
 4. A phenothiazine according to claim 1 wherein R^(5a) is benzyl or substituted benzyl.
 5. A pharmaceutical composition comprising a pharmaceutical grade carrier and at least one compound of any one of claims 1, 2, 3, or
 4. 6. The pharmaceutical composition of claim 5 wherein the carrier is for oral administration. 